The present invention relates to compositions and methods for detecting apoptosis or programmed cell death.
Throughout this disclosure, various publications are referenced by first author and date, within parentheses, patent number or publication number. The complete bibliographic reference is given at the end of the application for several of the references. The disclosures of these publications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this application pertains.
Apoptosis, also called programmed cell death (“PCD”), is a very important cellular process that plays a vital role in maintaining the normal physiological function of the organism [1,2]. For example, when DNA is damaged in a cell and cannot be repaired, the cell will enter apoptosis to avoid the formation of abnormalities in the tissue. In addition, the process of apoptosis is used in the thymus to eliminate self-reactive T cells to avoid auto-immunity. Apoptosis must be precisely regulated in order to maintain the proper functioning of our body. For example, failure to activate apoptosis can cause cancer or auto-immune diseases [3]. On the other hand, excessive activity of apoptosis can cause great damage to the organism; it can lead to many neurodegenerative diseases such as Huntington's disease and Alzheimer's disease [4, 5].
Since many important diseases, including cancer, AIDS, auto-immune diseases and neurodegenerative diseases are related to defective or excessive programmed cell death, drugs that can either facilitate or block programmed cell death can both be potentially useful in treating many diseases. Thus, there is a great interest in studying the process of apoptosis on a molecular basis. The signaling pathways that direct the programmed cell death process are very complicated [6-8]. Many external signals can trigger the initiation of apoptosis, including UV-irradiation, activation of the “death domain” via the TNF (tumor necrosis factor) receptor, treatment of hormone (e.g., glucocorticoid) or chemotherapy drugs (e.g., camptothecin) [6-9]. As for the internal signals, it is known that apoptosis is the outcome of a programmed cascade of intracellular events, which are centered on the activation of a class of cysteine proteases called “caspases” [7, 10]. Caspases are death proteases that are homologous to each other [22]. They are highly conserved through evolution and can be found from humans to insect, nematodes and hydra. [23-25]. Over a dozen caspases have been identified in humans, two thirds of which are believed to function in apoptosis [25, 26].
All known caspases possess an active-site cysteine and cleave substrates at Asp-Xxx bonds. The distinct specificity of each caspase is determined by the four residues amino terminus to the cleavage site [27]. Caspases are divided into subfamilies based on substrate preference, extent of sequence identity and structural similarities. For a review of the biochemistry of apoptosis and the role of caspases, see Hengartner, M. (2000) Nature 407:770-776.
At present, there exist a variety of techniques that can detect the process of apoptosis at different stages. For example, the terminal stage of apoptosis can be assayed by morphological changes of the cell (such as the presence of apoptotic bodies). Before that, apoptosis can be assayed by DNA fragmentation using either gel analysis or the TUNEL technique [11]. Early stages of apoptosis can be assayed by the turnover of PS (phosphatidylserine) in the membrane using an Annexin V-FITC labeled protein [12], or by detecting the activation of caspase-3 using a fluorescent dye linking to a substrate peptide [13]. All of these techniques, however, have certain limitations. For example, gel analysis can only be applied to an extract of cells, not to a single cell or intact cells. The TUNEL method can only be applied to fixed celled, not living cells. Annexin V can only detect events at the outer cell surface, not inside the cell. The caspase probe using a peptide linked fluorescence dye also has its own limitations. First, this probe cannot penetrate the cell membrane, and thus, it is typically used to assay cell extract. It is not an in vivo assay. Secondly, the fluorescent change resulting from caspase cleavage involves mainly a shift of the emission spectrum in the dye rather than a total destruction of the fluorescence. Its sensitivity is limited. Thus, a need exists for efficient and accurate compositions and methods to detect programmed cell death. This invention satisfies this need and provides related advantages as well.